Assignment of functional amino acids around the active site of human DNA topoisomerase IIα

An expression library for active site mutants of human topoisomerase IIα (TOP2α) was constructed by replacing the sequence encoding residues 793-808 with a randomized oligonucleotide cassette. This plasmid library was transformed into a temperature-sensitive yeast strain (top2-1), and viable transformants were selected at the restrictive temperature. Among the active TOP2α mutants, no substitution was allowed at Tyr⁸⁰⁵, the 5' anchor of the cleaved DNA, and only conservative substitutions were allowed at Leu⁷⁹⁴, Asp⁷⁹⁷, Ala⁸⁰¹, and Arg⁸⁰⁴. Thus, these 5 residues are critical for human TOP2α activity, and the remaining mutagenized residues are less critical for function. Using the x-ray crystal structure of yeast TOP2 as a structural model, it can be deduced that these 5 functionally important residues lie in a plane. One of the possible functions of this plane may be that it interacts with the DNA substrate upon catalysis. The side chains of Ser⁸⁰³ and Lys⁷⁹⁸, which confer drug resistance, lie adjacent to this plane.